Cryo-EM Reveals IMP Dehydrogenase I Filaments Adopt Diverse Architectures

To study the regulation of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanine nucleotide biosynthesis, we examined the effects of nucleosides, nucleotides, nucleotide analogs, or the IMPDH inhibitor mycophenolic acid (MPA) on the steady-state levels of IMPDH mRNA. The results indicated that IMPDH gene expression is regulated inversely by the intracellular level of guanine ribonucleotides. We have shown that treatment with guanosine increased the level of cellular guanine ribonucleotides and subsequently reduced IMPDH steady-state mRNA levels in a time- and dose-dependent manner. Conversely, MPA treatment diminished the level of guanine ribonucleotides and increased IMPDH mRNA levels. Both of these effects on the steady-state level of IMPDH mRNA could be negated by cotreatment with guanosine and MPA. The down regulation of IMPDH gene expression by guanosine or its up regulation by MPA was not due to major changes in transcriptional initiation and elongation or mRNA stability in the cytoplasm but rather was due to alterations in the levels of the IMPDH mRNA in the nucleus. These results suggest that IMPDH gene expression is regulated by a posttranscriptional, nuclear event in response to fluctuations in the intracellular level of guanine ribonucleotides.

Download Full-text

Active-site modification of native and mutant forms of inosine 5′-monophosphate dehydrogenase from Escherichia coli K12

Biochemical Journal ◽

10.1042/bj1910533 ◽

1980 ◽

Vol 191 (2) ◽

pp. 533-541 ◽

Cited By ~ 9

Author(s):

Harry J. Gilbert ◽

William T. Drabble

Keyword(s):

Escherichia Coli ◽

Binding Site ◽

Enzyme Inactivation ◽

Enzyme Hydrolysis ◽

Thiol Groups ◽

Imp Dehydrogenase ◽

Nitrobenzoic Acid ◽

Saturation Kinetics ◽

Mutant Forms ◽

Hydrolysis Of

IMP dehydrogenase of Escherichia coli was irreversibly inactivated by Cl-IMP (6-chloro-9-β-d-ribofuranosylpurine 5′-phosphate, 6-chloropurine ribotide). The inactivation reaction showed saturation kinetics. 6-Chloropurine riboside did not inactivate the enzyme. Inactivation by Cl-IMP was retarded by ligands that bind at the IMP-binding site. Their effectiveness was IMP>XMP>GMP»AMP. NAD+ did not protect the enzyme from modification. Inactivation of IMP dehydrogenase was accompanied by a change in λmax. of Cl-IMP from 263 to 290nm, indicating formation of a 6-alkylmercaptopurine nucleotide. The spectrum of 6-chloropurine riboside was not changed by IMP dehydrogenase. With excess Cl-IMP the increase in A290 with time was first-order. Thus it appears that Cl-IMP reacts with only one species of thiol at the IMP-binding site of the enzyme: 2–3mol of Cl-IMP were bound per mol of IMP dehydrogenase tetramer. Of ten mutant enzymes from guaB strains, six reacted with Cl-IMP at a rate similar to that for the native enzyme. The interaction was retarded by IMP. None of the mutant enzymes reacted with 6-chloropurine riboside. 5,5′-Dithiobis-(2-nitrobenzoic acid), iodoacetate, iodoacetamide and methyl methanethiosulphonate also inactivated IMP dehydrogenase. Reduced glutathione re-activated the methanethiolated enzyme, and 2-mercaptoethanol re-activated the enzyme modified by Cl-IMP. IMP did not affect the rate of re-activation of methanethiolated enzyme. Protective modification indicates that Cl-IMP, methyl methanethiosulphonate and iodoacetamide react with the same thiol groups in the enzyme. This is also suggested by the low incorporation of iodo[14C]acetamide into Cl-IMP-modified enzyme. Hydrolysis of enzyme inactivated by iodo[14C]acetamide revealed radioactivity only in S-carboxymethylcysteine. The use of Cl-IMP as a probe for the IMP-binding site of enzymes from guaB mutants is discussed, together with the possible function of the essential thiol groups.

Download Full-text